Spatiotemporal investigations of DNA damage repair using microbeams

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organisation is still unclear despite its decisive role in determining the fate of the damaged cell. Revealing the dynamic sequence of the repair proteins is therefore critical in understanding how the DNA repair mechanisms work. There are also still open questions regarding the possible movement of damaged chromatin domains and its role as trigger for lesion recognition and signalling in the DNA repair context. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. We have followed the development of radiation-induced foci for three DNA damage markers (i.e. γ-H2AX, 53BP1 and hSSB1) using normal fibroblasts (AG01522), human breast adenocarcinoma cells (MCF7) and human fibrosarcoma cells (HT1080) stably transfected with yellow fluorescent protein fusion proteins following irradiation with the QUB X-ray microbeam (carbon X-rays <2 µm spot). The size and intensity of the foci has been analysed as a function of dose and time post-irradiation to investigate the dynamics of the above-mentioned DNA repair processes and monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns—they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

Involvement of Exo1b in DNA damage-induced apoptosis

Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with singleand double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 A° in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short b-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Alternative dispute resolution : are apology and forgiveness simply two sides of the one coin?

Alternative dispute resolution (a.d.r.) processes are entrenched in western style legal systems. Forms of dispute resolution are utilised within schools and health systems; built in to commercial contracts; found in workplaces, clubs and organisations; and accepted in general day-to-day community disputes. The a.d.r. literature includes references to ‘apology’, but is largely silent on ‘forgiveness’. Where an apology is offered as part of a dispute resolution process, practice suggests that formalised ‘forgiveness’ rarely follows. Mediators may agree there is a meaningful place for apology in dispute resolution processes, but are most unlikely to support a view that forgiveness, as a conscious act, has an equivalent place. Yet, if forgiveness is not limited to the ‘pardoning of an offence’, but includes a ‘giving up of resentment’, or the relinquishing of a grudge, then forgiveness may play an underestimated role in dispute management. In the context of some day-to-day dispute management practice, this paper questions whether forgiveness should follow an apology; and concludes that meaningful resolutions can be reached without any formal element of ‘forgiveness’ or absolution. However, dispute management practitioners need to be aware of the latent role other aspects of forgiveness may play for the disputing parties.

An efficient approach for the direct synthesis of lithium niobate powders

Lithium niobate powders from the raw powders of Li2 O5 are directly synthesized by a combustion method with urea fuel. The synthesis parameters (e.g. the calcination temperature, calcination time, and urea-to-(Li2 CO3 + Nb2 O5) quantity ratio) are studied to reveal the optimized synthesis conditions for preparing high-quality lithium niobate powders. In our present work, it is found that a urea-to-(Li2 CO3 + Nb2 O5) ratio close to 3, calcination temperature at 550-600 degrees and reaction time around 2.5h may lead to high-quality lithium niobate powsers. The microstructure of synthesized powders is further studied; a possible mechanism of the involved reactions is also proposed.

Sustaining the next generation of teacher-researchers to work for social justice

This chapter explores a research project involving teachers working with some of the most disadvantaged young people in South Australia, children growing up in poverty, in families struggling with homelessness and ill-health, in the outer southern suburbs. Additionally, there were particular children were struggling with intellectual, emotional and social difficulties which were extreme enough for them not be included in a mainstream class. The research project made two crucial interrelated moves to support teachers to tackle this tough work. First, the project had an explicit social justice agenda. We were not simply researching literacy outcomes, but literacy pedagogies for the students teachers were most worried about. And we wanted to understand how the material conditions of students’ everyday lifeworlds impacted on the working conditions of teachers’ schoolworlds. We sought to open up a discursive space where teachers could talk about poverty, violence, racism and classism in ways that would take them beyond despair and into new imaginings and positive action. Second, the project was designed to start from the urgent questions of early career teachers and to draw on the accumulated practice wisdom of their chosen mentors. Hence we designed not only a teacher-researcher community, but cross-generational networks. Our aim was to build the capacities of both generations to address long-standing educational problems in new ways that drew overtly on their different and complementary resources.

Periodontal regeneration and mesenchymal stem cells

The ultimate goal of periodontal therapy is to regenerate periodontal supporting tissues, but this is hard to achieve as the results of periodontal techniques for regeneration are clinically unpredictable. Stem cells owing to their plasticity and proliferation potential provides a new paradigm for periodontal regeneration. Stem cells from mesenchyme can self renew and generate new dental tissues (including dentin and cementum), alveolar bone and periodontal ligament, and thus they have great potential in periodontal regeneration. This chapter presents an insight into mesenchymal stem cells and their potential use in periodontal regeneration. In this chapter the cellular and molecular biology in periodontal regeneration will be introduced, followed by a range of conventional surgical procedures for periodontal regeneration will be discussed. Mesenchymal stem cells applied in regenerated periodontal tissue and their biological characterizations in vitro will be also introduced. Lastly, the use of mesenchymal stem cell to repair periodontal tissues in large animal models will be also reviewed.

Changes in resting and walking energy expenditure and walking speed during pregnancy in obese women

Background Energy conserving processes reported in undernourished women during pregnancy are a recognised strategy to provide energy required to support fetal development. Women who are obese before conceiving arguably have sufficient fat stores to support the energy demands of pregnancy without the need to provoke energy conserving mechanisms. Objective We tested the hypothesis that obese women would demonstrate behavioural adaptation (i.e. decrease in self-selected walking (SSW) speed) but not metabolic compensation (i.e. decrease in resting metabolic rate (RMR) or metabolic cost of walking) during gestation. Design RMR, SSW speed, metabolic cost of walking, and anthropometry were measured in 23 women (BMI: 33.6 ± 2.5 kg/m2; 31 ± 4 years) at approximately weeks 15 (wk 15) and 30 (wk 30) of gestation. RMR was also measured in two cohorts of non-pregnant controls matched for age, weight and height of the pregnant cohort at wk 15 (N=23) and wk 30 (N=23). Results GWG varied widely (11.3 ± 5.4 kg) and 52% of women gained more weight than is recommended. RMR increased significantly by an average 177 ± 176 kcal/d (11±12%; P<0.0001); however the within-group variability was large. Both the metabolic cost of walking and SSW speed decreased significantly (P<0.01). While RMR increased in >80% of the cohort, the net oxygen cost of walking decreased in the same proportion of women. Conclusions While the increase in RMR was greater than was explained by weight gain, there was evidence of both behavioural and biological compensation in the metabolic cost of walking in obese women during gestation.

Determinants and consequences of information sharing with key suppliers

The economic environment of today can be characterized as highly dynamic and competitive if not being in a constant flux. Globalization and the Information Technology (IT) revolution are perhaps the main contributing factors to this observation. While companies have to some extent adapted to the current business environment, new pressures such as the recent increase in environmental awareness and its likely effects on regulations are underway. Hence, in the light of market and competitive pressures, companies must constantly evaluate and if necessary update their strategies to sustain and increase the value they create for shareholders (Hunt and Morgan, 1995; Christopher and Towill, 2002). One way to create greater value is to become more efficient in producing and delivering goods and services to customers, which can lead to a strategy known as cost leadership (Porter, 1980). Even though Porter (1996) notes that in the long run cost leadership may not be a sufficient strategy for competitive advantage, operational efficiency is certainly necessary and should therefore be on the agenda of every company. ----- ----- ----- Better workflow management, technology, and resource utilization can lead to greater internal operational efficiency, which explains why, for example, many companies have recently adopted Enterprise Resource Planning (ERP) Systems: integrated softwares that streamline business processes. However, as today more and more companies are approaching internal operational excellence, the focus for finding inefficiencies and cost saving opportunities is moving beyond the boundaries of the firm. Today many firms in the supply chain are engaging in collaborative relationships with customers, suppliers, and third parties (services) in an attempt to cut down on costs related to for example, inventory, production, as well as to facilitate synergies. Thus, recent years have witnessed fluidity and blurring regarding organizational boundaries (Coad and Cullen, 2006). ----- ----- ----- The Information Technology (IT) revolution of the late 1990’s has played an important role in bringing organizations closer together. In their efforts to become more efficient, companies first integrated their information systems to speed up transactions such as ordering and billing. Later collaboration on a multidimensional scale including logistics, production, and Research & Development became evident as companies expected substantial benefits from collaboration. However, one could also argue that the recent popularity of the concepts falling under Supply Chain Management (SCM) such as Vendor Managed Inventory, Collaborative Planning, Replenishment, and Forecasting owe to the marketing efforts of software vendors and consultants who provide these solutions. Nevertheless, reports from professional organizations as well as academia indicate that the trend towards interorganizational collaboration is gaining wider ground. For example, the ARC Advisory Group, a research organization on supply chain solutions, estimated that the market for SCM, which includes various kinds of collaboration tools and related services, is going to grow at an annual rate of 7.4% during the years 2004-2008, reaching to $7.4 billion in 2008 (Engineeringtalk 2004).

Time course of temperature effects on cardiovascular mortality in Brisbane, Australia

Objective To quantify the lagged effects of mean temperature on deaths from cardiovascular diseases in Brisbane, Australia. Design Polynomial distributed lag models were used to assess the percentage increase in mortality up to 30 days associated with an increase (or decrease) of 1°C above (or below) the threshold temperature. Setting Brisbane, Australia. Patients 22 805 cardiovascular deaths registered between 1996 and 2004. Main outcome measures Deaths from cardiovascular diseases. Results The results show a longer lagged effect in cold days and a shorter lagged effect in hot days. For the hot effect, a statistically significant association was observed only for lag 0–1 days. The percentage increase in mortality was found to be 3.7% (95% CI 0.4% to 7.1%) for people aged ≥65 years and 3.5% (95% CI 0.4% to 6.7%) for all ages associated with an increase of 1°C above the threshold temperature of 24°C. For the cold effect, a significant effect of temperature was found for 10–15 lag days. The percentage estimates for older people and all ages were 3.1% (95% CI 0.7% to 5.7%) and 2.8% (95% CI 0.5% to 5.1%), respectively, with a decrease of 1°C below the threshold temperature of 24°C. Conclusions The lagged effects lasted longer for cold temperatures but were apparently shorter for hot temperatures. There was no substantial difference in the lag effect of temperature on mortality between all ages and those aged ≥65 years in Brisbane, Australia.